Advances in research of interchangeable immunization with live attenuated Japanese encephalitis vaccines and inactivated vaccines / 中华预防医学杂志

Japanese encephalitis (JE) virus is the leading cause of vaccine-preventable encephalitis in Asia and the Western Pacific, which mainly invades central nervous system. Vaccination is the most important strategy to prevent JE. Currently, both live attenuated Japanese encephalitis vaccines (JE-L) and inactivated vaccines (JE-I) are in use. Due to the supply of vaccines and the personal choice of recipients, there will be a demand for interchangeable immunization of these two vaccines. However, relevant research is limited. By reviewing domestic and foreign research evidence, this article summarizes the current situation of the interchangeable use of JE-L and JE-I, and makes recommendations when the interchangeable immunization is in urgent need, so as to provide reference for practical vaccination and policymaking in China.

A Centralized Report on Pediatric Japanese Encephalitis Cases from Beijing Children's Hospital, 2013 / 生物医学与环境科学（英文）

Fifteen pediatric cases of suspected Japanese encephalitis (JE) were reported in Beijing Children's Hospital during the late summer of 2013. The clinical manifestations in most cases included high fever, seizures, and abnormal magnetic resonance imaging findings. Twelve of 15 cases were laboratory-confirmed as JE cases by pathogen identification. Epidemiological investigations showed that five of the 12 laboratory-confirmed patients had an incomplete JE vaccination history. Follow-up investigations after discharge indicated that seven laboratory-confirmed JE patients without JE vaccinations had relatively poor prognoses, with an average Modified Rankin Scale (MRS) score of 2.6 when compared with the other five laboratory-confirmed, JE-vaccinated patients with an average MRS score of 0.5. The observation of pediatric JE cases among those with a history of JE vaccination warrants further attention.

Generation of Japanese Encephalitis Virus-like Particle Vaccine and Preliminary Evaluation of Its Protective Efficiency / 病毒学报

The cDNA fragment of JEV prME gene was cloned into the baculovirus shuttle vector (bacmid) to construct a recombinant baculovirus vector, defined as AcBac-prME. Then the recombinant baculovirus Ac-prME was obtained by transfecting Sf9 cells with AcBac-prME. Western blot analysis and immunofluorescence results indicated that both prM and E proteins were efficiently expressed in Sf9 cells. Electron microscopy suggested that prME was assembled into JEV-VLPs. To further evaluate the potential of JEV-VLPs as vaccine, the mice were immunized with JEV-VLPs and then challenged with lethal JEV. The results of mice survival and pathological changes demonstrated that the JEV-VLPs performed complete protection against JEV-P3 strain and relieved pathological changes in the mice brain significant. This study suggest that JEV-VLPs would be a potential vaccine for Japanese encephalitis virus.

The Immunogenicity and Safety of the Live-attenuated SA 14-14-2 Japanese Encephalitis Vaccine Given with a Two-dose Primary Schedule in Children

Effective and tolerable vaccination is an essential strategy to prevent Japanese encephalitis (JE) in endemic areas. Although the live attenuated SA 14-14-2 JE vaccine (LAJEV) has been widely used since its introduction, the systemic data of LAJEV was very rarely available in Korea. We conducted the open-label, prospective cohort study to assess the immunogenicity and safety of this vaccine. Ninety subjects were enrolled, and LAJEV in a 2-dose primary series was given with a 12-month interval. Neutralizing antibody titers were measured before and after each vaccination, and active monitoring for adverse events was performed. After the first dose, 91.1% of subjects had seroprotection with a geometric mean titer (GMT) of 40.9. Seroprotection rate after the second dose was 97%, and GMT showed an increase of 6.5-fold. Most adverse events following immunization were self-limited, and no serious adverse events were reported until 42 days after each dose. The 2-dose administration of LAJEV in the primary immunization schedule appeared to be highly immunogenic and safe.

Effects of cell-mediated immunity induced by intramuscular chitosan-pJME/ GM-CSF nano-DNA vaccine in BAlb/c mice / 病毒学报

This study aimed to investigate the immune adjuvant effect and mechanism induced by chitosan nanoparticles carrying pJME/GM-CSF. In this study, plasmid DNA (pJME/GM-CSF) was encapsulated in chitosan to prepare chitosan-pJME/GM-CSF nanoparticles using a complex coacervation process. Immunohistochemistry was used to detect the type of infiltrating cells at the site of intramuscular injection. The phenotype and functional changes of splenic DCs were measured by flow cytometry after different immunogens were injected intramuscularly. The killing activity of CTLs was assessed using the lactate dehydrogenase (LDH) release assay. The preparation of chitosan-pJME/GM-CSF nanoparticles matched the expected theoretical results. Our results also found that, after pJME/GM-CSF injection, the incoming cells were a mixture of macrophages, neutrophils, and immature DCs. Meanwhile, pJME/GM-CSF increased the expression of MHC class II molecules on splenic DCs, and enhanced their Ag capture and presentation functions. Cell-mediated immunity was induced by the vaccine. Furthermore, chitosan-pJME/GM-CSF nanoparticles outperformed the administration of standard pJME/GM-CSF in terms of DC recruitment, antigen processing and presentation, and vaccine enhancement. These findings reveal that chitosan could be used as delivery vector for DNA vaccine intramuscular immunizations, and enhance pJME/GM-CSF-induced cellular immune responses.

Progress in the research of phenotype and genotype of Japanese encephalitis virus in China / 病毒学报

Japanese encephalitis virus(JEV)is one of the leading cause of viral encephalitis in Asia. The phenotypic and genotypic characteristics of isolated virus strains are reviewed in this paper. Studies on the biological characteristics of the isolates showed that different isolates existed apparent differences in virus plaque morphology, neuroinvasive pathogenicity in mice, protective antigenicity and hemagglutination property. In China, only genotype III JEV strains were isolated before 1977. But since 1977, both genotype I and I JEV strains were isolated and the genotype I virus, which was isolated from mosquitoes mostly, has become the dominant strain. Study on the genomic sequence indicated that there was only a few amino acid difference (< or = 43%) between the two genotype isolates. Comparison between both genotype isolates and widely used live vaccine strain SA14-14-2 revealed that there were only < or = 3% amino acid differences, most of which were the SA14-14-2 unique attenuating sites. These results indicate that the SA14-14-2 live vaccine is able to protect people against infection of the both genotype I and Ill JEV strains.

Japanese encephalitis: a review of the Indian perspective

Japanese encephalitis virus (JEV) causes Japanese encephalitis, which is a leading form of viral encephalitis in Asia, with around 50,000 cases and 10,000 deaths per year in children below 15 years of age. The JEV has shown a tendency to extend to other geographic regions. Case fatality averages 30% and a high percentage of the survivors are left with permanent neuropsychiatric sequelae. Currently, there is no cure for JEV, and treatment is mainly supportive. Patients are not infectious, but should avoid further mosquito bites. A number of antiviral agents have been investigated; however, none of these have convincingly been shown to improve the outcome of JEV. In this review, the current knowledge of the epidemiology and the pathogenesis of this deadly disease have been summarized.

Molecular characteristics of the genome of G I of Japanese encephalitis virus isolated from the specimen collected from viral encephalitis case for the first time / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the molecular basis of pathogenicity of Japanese encephalitis virus (JEV) by sequencing of complete nucleotide sequence and analyze the characteristics of full-length genome of genotype I Japanese encephalitis virus strains (GZ56) which was isolated from the first cerebrospinal fluid (CSF) of Japanese encephalitis patients.</p><p><b>METHODS</b>The complete nucleotide sequence was obtained by RT-PCR and sequencing was performed directly. Bioinformatics was used to analyze the nucleic acid data, deduced amino acid sequence and phylogenetic trees.</p><p><b>RESULTS</b>The result of sequence analysis showed that the genome of GZ56 strains had 10 965 nucleotides, which coded for a 3432-amino acid polyprotein. Phyolngenetic analysis based on full-length genome showed that GZ56 strains and M-28 strains which were the first isolated from mosquitoes in Yunnan in 1977 were in the same evolutionary branch. GZ56 strains belongs to genotype I of Japanese encephalitis virus, the homology of genome ranged from 96.2% to 98.6% in nucleotide and from 98.2% to 99.7% in amino acid sequences respectively when compared with selected genotype I of JEV strains in GenBank. There were 11 amino acid divergences in E protein when compared with the JEV inactivated P3 strain but they are not the key virulence sites. However, there were 14 amino acid divergences in E protein when compared with the JEV live attenuated vaccine SA14-14-2 strain and 8 amino acid divergences were the key virulence sites.</p><p><b>CONCLUSION</b>This study indicated that the full length of genome GZ56 strains had no ignificant change. It can be hypothesized from genomic level that the currently available JEV vaccines(inactivated and live attenuated) can protect against GZ56 strains infection, meanwhile, the JEV live attenuated vaccine (SA14-14-2) formulation conferred higher levels of protection.</p>

A survey of Japanese encephalitis antibody migrant workers in Shenzhen 2009 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To understand the immunological status of Japanese encephalitis (JE) antibodies amongst migrant workers and to provide epidemiological basis for public health strategies on JE prevention and control in Shenzhen.</p><p><b>METHODS</b>A multi-stage random sampling method was used, and 1003 migrant workers aged 18 to 60 from 44 factories were investigated and their serum specimens were collected. The enzyme-linked immunosorbent assay (ELISA) was used to detect JE antibodies qualitatively.</p><p><b>RESULTS</b>The gross IgG seroprevalence rate for JE was 20.2% (203/1003). Sex-specified seroprevalence was 21.2% (103/485) for male and 19.3% (100/518) for female, respectively (χ(2) = 579, P > 0.05). Age-specific seropositive rates were 22.6% (12/53) for those below 20 years old, 18.7% (120/642) for those between 20-years old, 26.0% (58/223) for those between 30-years old and 15.3% (13/85) for those on or above 40 years old (χ(2) = 7.96, P > 0.05). Proportions for self-reported positive immunization, non-immunization and unclear immunization history were 22.1% (30/136), 22.1% (51/231) and 19.2% (122/636), respectively (χ(2) = 501, P > 0.05). Seroprevalence by region of origins showed that workers from Guangdong province was the highest (30.5%, 50/164), followed by workers from Guangxi (29.7%, 22/74) whilst workers from Shan(3)xi (5.4%, 2/37) had the lowest rate. Seroprevalence rate for managers (29.0%, 31/107) was higher than that of technicians (7.1%, 1/14) (χ(2) = 21.78, P < 0.05). Serological positive rate of workers with university or above educational background was the highest (32.7%, 16/49), followed by that for individuals with college degree (10.3%, 10/97) (χ(2) = 13.02, P < 0.05).</p><p><b>CONCLUSION</b>No associations are detected between JE seroprevalence and age, or sex, or self-reported immunization histories amongst migrant labor workers in Shenzhen. However, correlations between JE serological positive rate and region of origins, occupation and educational attainment are found to be significant. The gross seroprevalence of JE antibodies suggests that the level of JE antibodies amongst Shenzhen migrant workers is low and the population immunity barrier has yet to be established. It is necessary to strengthen prevention and control strategies of JE among labor workers of Shenzhen.</p>

Comparison of viremia formation between guinea-pigs infected with wild and attenuated (SA14-14-2) Japanese encephalitis viruses / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the viremia formation in guinea-pigs infected with wild type and attenuated Japanese encephalitis virus (JEV).</p><p><b>METHODS</b>Guniea pigs were inoculated intraperitoneally with different wild JEV strains and the attenuated vaccine strain and its parent virulent strain. Viremia was detected on different days following virus inoculation.</p><p><b>RESULTS</b>All the guinea-pigs inoculated with the wild JEV strains induced different levels of viremia (1.00-3.40 Lg pfu) on the 1st and 3rd day post inoculation. Using a virus titer of 10(4) pfu for inoculation, the animals inoculated with the SA14 parent strain induced relatively high viremia (10(2.4)-10(3.4) pfu), however no viremia coulds be detected on any tested days.</p><p><b>CONCLUSION</b>The degree of viremia in guinea pigs can be used as a new method to evaluate the attenuation of JEV.</p>

Investigation on the Frequency and Severity of Common Adverse Reactions of Japanese Encephalitis Vaccines / 소아감염

PURPOSE: To evaluate the number and severity of adverse reactions after Japanese Encephalitis (JE) vaccination in children using different vaccines (inactivated vaccine or live attenuated vaccine) and to determine the ability and safety of the vaccines to provide effective immunization for JE. METHODS: From August 2006 to February 2007, we conducted a prospective cohort study of the adverse reactions associated with JE immunization in Korea. We investigated common adverse reactions during the 4 days following immunization using telephone collaborations with four public health centers and nine pediatric clinics. RESULTS: The mean age of children receiving the inactivated vaccines and live attenuated vaccines, respectively, were 1.4 y (range: 1 to 8.5) and 1.7 y (range: 1 to 8.3). The number of children that received the inactivated vaccines was 425 (64.6%). A total of 233 (35.4%) received the live attenuated vaccines. Fourteen children (3.3%) had more than one localized adverse event with the inactivated vaccine, and six (2.6%) had more than one event with the live attenuated vaccine (P=0.607). Systemic adverse reactions occurred in 5.2% vs. 8.2%, respectively, of these groups (P=0.131). Fever was more common in the live attenuated vaccine group than in the inactivated vaccine group on the day of vaccination (P=0.026). CONCLUSIONS: The rate of adverse events in our study was even lower than that previously reported. No significant difference in outcomes between inactivated vaccine and live attenuated vaccine was found in JE-immunized children. Fever was more common in the live attenuated vaccine group than in the inactivated vaccine group on the day of vaccination.

Immunogenicity and antigenicity of Japanese encephalitis virus envelope protein domain III / 生物工程学报

To express the domain III gene of Japanese encephalitis virus (JEV) and to learn the possibility of developing the Dil protein as a subunit vaccine, we amplified the JEV DIII gene by PCR and constructed the expression plasmid pET-JE DIII by inserting JEV DIII gene into the prokaryotic expression vector pET-32a(+). The domain III protein of the attenuated strain SA14-14-2 was expressed as a thioredoxin (Trx) fusion protein, which was unique in forming a large fraction of the soluble recombinant protein. We immunized the rabbits and mice with the purified protein, tested the antigenicity and immunogenicity of JEV DIII protein by ELISA, Western blotting, plaque reduction test and observed the protective efficacy on challenged weanling mice with JEV. Rabbits immunized with the purified JEV DIII protein generated 1: 7 x 10(5) anti-JEV specific antibody titers. BALB/c mice immunized with the purified JEV E DIII protein generated 1: 8.2 x 10(4) anti-JEV specific antibody titers. And the neutralized antibody titer can reach 1:256, the survival rate of the immunized weanling mice was approximately 75%. Overall, this study highlighted that recombinant JEV E DIII protein delivered in mice and rabbits can generate high antibody titers against JEV, and protect some mice challenged with JEV. These studies can provide useful information for further developing the domain III recombinant protein as subunit vaccine against JEV.

Bellary, India achieves negligible case fatality due to Japanese encephalitis despite no vaccination: an outbreak investigation in 2004.

OBJECTIVE: To confirm the existence of the outbreak of suspected Japanese encephalitis, identify the source, to understand the circumstances due to which the outbreak was taking place and to suggest measures for its control. METHODS: The team visited Bellary from 4th to 10th Sept, 2004. The team interviewed the key persons and analyzed the records at District Surveillance Unit and Entomological Surveillance Unit and case records of suspected JE cases admitted in Encephalitis ward in Vijay Nagar Institute of Medical Sciences (VIMS). Eco-entomological survey was done in houses and surroundings of 3 randomly selected cases of Encephalitis in rural and urban areas of District Bellary. Their family members and neighbors were also asked for the awareness and presence of disease. Data was analyzed for epidemiological and clinical profiles. RESULTS: The suspected JE cases were being reported from end of June 2004. The cases were sporadic and out of 34 cases reported to VIMS (till 10th of September), 32 were from Bellary district and 2 were from adjoining Andhra Pradesh. Among these 32, 22 were from Bellary Taluk, which in turn were mainly concentrated (10 were reported) in urban Bellary. The case fatality rate was zero as no death was reported. Entomological surveillance (done by District Surveillance Unit) revealed a high outdoor presence of Culex tritaeniorhynchus as well as an indoor rising density of this mosquito from 2 per man hour catch in January to 22 in the month of August in the affected villages. On the contrary, the investigations on 7th and 8th September revealed high densities of An.subpictus and An. peditaenatus and nil of Culex species in the urban areas. Amplifier host of pigs and water birds were occasionally sighted in the area. CONCLUSION: A good community awareness of encephalitis, a prompt referral system and a good supportive treatment for the patients and a good surveillance system and response were observed. Very close proximity with amplifying hosts of pigs was avoided by the community, though piggeries were still not very far away (1-3 Km). These may explain the reduction in cases, deaths and disabilities due to this disease in this district over the years. Possibilities of mutant strain which is less virulent and/or a better immune status of at risk population may also need to be explored. The impact of the mass vaccination with SA 14-14-2, imported from China in Bellary during July, 2006 remains to be evaluated. This will further decrease the case load.

Epidemiological situation of Japanese encephalitis in Nepal.

A human Japanese encephalitis (JE) case is considered to have elevated temperature (over 380 C) along with altered consciousness or unconsciousness and is generally confirmed serologically by finding of specific anti-JE IgM in the cerebro spinal fluid. No specific treatment for JE is available. Only supportive treatment like meticulous nursing care, introduction of Ryle's tube if the patient is unconscious, dextrose solution if dehydration is present, manitol injection in case of raised cranial temperature and diazepam in case of convulsion. Intra venous fluids, indwelling catheter in conscious patient and corticosteroids unless indicated should be avoided. Pigs, wading birds and ducks have been incriminated as important vertebrate amplifying hosts for JE virus due to viremia in them. Man along with bovines, ovines and caprines is involved in transmission cycle as accidental hosts and plays no role in perpetuating the virus due to the lack of viremia in them. The species Cx tritaeniorhyncus is suspected to be the principal vector of JE in Nepal as the species is abundantly found in the rice-field ecosystem of the endemic areas during the transmission season and JE virus isolates have been obtained from a pool of Cx tritaeniorhyncus females. Mosquito vector become infective 14 days after acquiring the JR virus from the viremic host. The disease was first recorded in Nepal in 1978 as an epidemic in Rupandehi district of the Western Development Region (WDR) and Morang of the Eastern Region (EDR). At present the disease is endemic in 24 districts. Although JE as found endemic mainly in tropical climate areas, existence and proliferation of encephalitis causing viruses in temperate and cold climates of hills and valleys are possible. Total of 26,667 cases and 5,381 deaths have been reported with average case fatality rate of 20.2% in an aggregate since 1978. More than 50% of morbidity and 60% mortality occur in the age group below 15 years. Upsurge of cases take place after the rainy season (monsoon). Cases start to appear in the month of April - May and reach its peak during late August to early September and start to decline from October. There are four designated referral laboratories, namely National Public Health Laboratory (Teku), Vector Borne Diseases Research and Training Center (Hetauda), B.P. Koirala Institute of Medical Sciences (Dharan) and JE Laboratory (Nepalgunj), for confirmatory diagnosis of JE. For prevention of JE infection; chemical and biological control of vectors including environmental management at breeding sites are necessary. Segregate pigs from humans habitation. Wear long sleeved clothes and trousers and use repellent and bed net to avoid exposure to mosquitos. For the prevention of the disease in humans, safe and efficacious vaccines are available. Therefore immunize population at risk against JE. Immunize pigs at the surroundings against JE. 225,000 doses of live attenuated SA-14-14.2 JE vaccine were received in donation from Boran Pharmaceuticals, South Korea for the first time in Nepal. Altogether 224,000 children aged between 1 to 15 years were vaccinated in Banke, Bardiya and Kailali districts during 1999. From China also, 2,000,000 doses of inactivated vaccine were received in 2000 and a total of 481,421 children aged between 6m to 10 yrs were protected from JE during 2001/2002. Ministry of Agriculture, Department of Livestock Services has vaccinated around 200,000 pigs against JE in terai zone during February 2001.

Construction of infectious Japanese encephalitis virus clone based on the cDNA template of the attenuated live vaccine production strain SA14-14-2 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct infectious Japanese encephalitis virus (JEV) based on the in vitro-ligated cDNA template of the vaccine strain SA14-14-2, and identify the virus.</p><p><b>METHODS</b>Full-length genomic cDNA of JEV SA14-14-2 strain was ligated and then RNA was transcribed in vitro, the infective virus was obtained by transfecting the RNA into Vero cells and identified.</p><p><b>RESULTS</b>The infective clone of JEV was constructed, the virulence was weaker than the wild virus.</p><p><b>CONCLUSION</b>It was possible to construct infectious clone from the production strain of live attenuated Japanese B encephalitis vaccine.</p>

Research on Culex tritaeniorhynchus and Culex pipiens quinquefasciatus intrathoracically infected with attenuated Japanese encephalitis virus SA14-14-2 vaccine strain / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To determine if the attenuated Japanese encephalitis (JE) virus SA14-14-2 vaccine strain interacts efficiently with Culex tritaeniorhynchus and Culex pipiens quinquefasciatus, and further to acquire a new knowledge of its characteristics and safety for human beings.</p><p><b>METHODS</b>Laboratory colonies of the two species of mosquitoes were set up and were inoculated intrathoracically with the attenuated vaccine virus and wild JE virus (Nak), both of which were used with different dilution from 10(-1) to 10(-9). Subsequently, the virus titers in the mosquitoes were detected by the plaque assay.</p><p><b>RESULTS</b>Inoculated with the vaccine strain, two species of mosquitoes were infected with the titers ranged from 10(0)-10(-3), and the maximum titers in Culex tritaeniorhynchus and Culex pipiens quinquefasciatus were 4.48 logPFU/ml and 5.63 logPFU/ml, respectively. Inoculated with wild JE virus, Culex pipiens quinquefasciatus was infected with titers ranged from 10(0)-10(-5), and the maximum titer in the mosquitoes was 6.59; Culex tritaeniorhynchus was infected with titers ranged from 10(0)-10(-4) and the maximum titer was 5.74 logPFU/ml.</p><p><b>CONCLUSION</b>By intrathoracic infection, the attenuated JE virus SA14-14-2 vaccine strain can replicate in both species of mosquitoes.</p>

Chemical components analysis and morphology observation of virus in Japanese encephalitis vaccine produced from Beijing strain

The first 5 lots of Japanese encephalitis vaccines produced from Beijing-1 strain: JB011203, JB021203, JB030104, JB040204 and JB050304 were controlled of quality on chemical components and virus morphology in vaccine, using reference vaccine EJP034A (BIKEN-Japan). The results showed that: TCA-protein was 13.2- 14.5 µg/ml, the reference was 11.1 µg/ml (WHO standard 5-40 µg/ml). The thimerosal content was 82-84 µg/ml, the reference 76,3 µg/ml (WHO standard ≤120 µg/ml). The formaldehyde content was 0.061-0.063 µg/ml, the reference was 2.19 µg/ml (WHO standard ≤100 µg/ml). pH was 7.02-7.04, the reference was 7.10 (WHO stand. 6.8-7.4). The viruses morphology after negative stain were observed on electromicroscope JEM1010 with enlarge x 90.000 and x 150.000. The identical ball form of purified viruses with the intact surface antigen. 5/5 lots were passed the minimum requirements of biological products of WHO.

Comparison of the infected mouse brain by Nakayama and Beijing -1 strain for Japanese Encephalitis vaccine production

Using white Swiss mouse at 3-4 weeks of age for injection of Japanese Encephalitis (JE) virus. Every 8 lots of the mouse had been infected with Nakayama and Beijing-1 strains by injection of 0.03ml into their brains, then followed up within 3 days. The paralysed mice were collected at the 69th hour of the beginning and at the 120th hour of the end. The interval between collection time was every 3 hours. In this study, the rate of mice with paralysis due to Beijing-1 strain was higher than those due to Nakayama strain (94.99% and 92.97%, respectively). The authors suggested that use of Beijing-1 strain to produce JE vaccine is more effective than Nakayama strain.

Evaluation of safety and potency of Japanese encephalitis vaccine produced by Beijing-1 strai

The pilot study was performed on the first 5 lots of the Japanese encephalitis vaccine produced by Beijing-1 strain: JB011203, JB021203, JB030104, JB040204 and JB050304 to evaluate their safety and potency. The quality control consists of abnormal toxicity, specific safety, pyrogen, sterility and potency test. All of 5 lots were passed the minimum requirements of biological products of Japan. The mice and guinea pigs were healthy, body weigh increased. Non of bacteria and fungi grown in both medium Thioglycholate and Soybeancasein. The pyrogen test on rabbits was passed in 5/5 lots. Summed temperature response of 3 rabbits per lot were 0°C-0.3°C (WHO requirement ≤1.4°C). Potency test was evaluated by PRNT compared with reference EJP034A of BIKEN. After the second immunization the neutralizing antibody response > reference from 0.04 to 0.2 log. 5/5 lots were passed the safety and potency test and met the minimum requirement of biological products of WHO.